Pharmaceutical Formulation of RNA
In addition to their central role as carriers of genetic information in the form of messenger RNA (mRNA), Ribonucleic Acid molecules (RNA) have recently been recognized to be pathogen-associated molecular patterns (PAMP) inducing immunostimulation (isRNA) and to be regulators of gene expression (antisense, small interfering RNA, siRNA and micro RNA, miRNA). All those natural activities of different types of naturally occurring RNA molecules can be reproduced using RNA produced in vitro (chemical or enzymatic synthesis) that are introduced in cells. Although exogenous naked nucleic acids (RNA or Deoxyribonucleic Acids, DNA) can be spontaneously taken up and biologically active in vivo [1], their activities are enhanced by methods (electroporation) or formulations (complexation by cationic polymers) aiming to favor their penetration in cells. Many reagents have been used to obtain such transfection of RNA molecules: cationic compounds like the peptide protamine [2] or polyethylenimine (PEI) [3, 4] or chitosan and/or lipophilic molecules such as cationic lipids that spontaneously form liposomes or micelles when mixed with nucleic acids in adequate conditions (recent review by Ozpolat et al. [5]). For clinical applications, those transfection reagents must be produced at pharmaceutical grade and mixed with RNA in a way that produces specified (size of particles, percentage of encapsulated RNA, etc.) formulations. The formulations may be more or less stable and toxic. Thus, alternatives which allow easy formulation of RNA for transfection of cells are needed. We here provide an invention and possible solution for this problem by demonstrating that RNA can penetrate cells, when it has an alkali metal (preferably sodium) as counter ion and when it is formulated in the presence of dication(s) (preferably calcium). Optimally, the RNA contains a poly-G (more than 2 consecutive G residues) or a poly-U (more than 4 consecutive U residues) or a GPurine(n)G (where Purine is G or A residues and n from 1 to 4 or more) sequence. Those penetrating RNAs can bring an attached cargo (chemical or biological entity) inside cells.
Immunostimulating RNA (isRNA)
Immunostimulatory nucleic acid molecules include DNA comprising the unmethylated CpG motif (CpG oligodeoxynucleotide: CpG ODN), RNA in the form of double-stranded RNA (dsRNA), and stabilized, protected or otherwise chemically modified single-stranded RNA molecules (ssRNAs). The different families of nucleic acid PAMPs (i.e. CpG ODN, dsRNA, and ssRNA) are known to trigger different intracellular (located in endosomes) Toll-like receptors (TLRs) expressed by non-overlapping immune cell populations [6]. Those receptors give rise to different types of innate immune responses characterized by the secretion of a specific panel of cytokines including, but not limited to, for example, interleukine-6, Tumor Necrosis Factor (TNF)-alpha, or interferon-alpha. DNA triggers TLR-9, whereas dsRNA triggers TLR-3, and ssRNA triggers TLR-7 as well as TLR-8 (recently reviewed by Panter et al. [7]). For ssRNA, it was reported that RNA oligonucleotides stimulate preliminary through their U residues [3, 4]. Because exogenous stabilized ssRNA activates the innate immunity, it can be used as an adjuvant for vaccines as described by Scheel et al. for naked phosphorothioate RNA oligonucleotides [8] and Bourquin et al. [9] for liposome encapsulated RNA oligonucleotides.
Dielbold et al. [10] further showed that, when delivered to endosomes, viral and self RNA triggered equally efficiently TLR7 mediated innate immune response, further supporting the notion that discrimination between self and viral RNA ligands is based on endosomal accessibility rather than RNA sequence. Thus immunostimulation by exogenous RNA eventually linked to an antigen depends on the penetration of those molecules inside the cells. Thereby, isRNA must be formulated in transfection reagents. This step complicates the development of pharmaceutical isRNA and raises stability as well as toxicity issues.
The solution to the above technical problem is provided by the embodiments of the present invention as defined in the claims.
Protein Coding RNA (mRNA)
Long RNA molecules constitute the genome of some viruses (e.g. Influenza virus, HIV) and the intermediate genetic information between DNA and protein in all living cells. Such mRNA can be extracted from cells or produced in vitro by enzymatic transcription of linearized DNA plasmids, purified and used to transfect cells (reviewed by Pascolo [11]). In vitro transfection is routinely made thanks to electroporation or RNA-encapsulation. In the first case, mRNA is introduced in the cells by a short electric pulse. In the second case, lipids, most often cationic lipids or cationic peptides or cationic sugars are used to encapsulate mRNA, allowing its delivery in cells. Once in cells, mRNA is translated in proteins. In vivo, the direct skin injection of mRNA results in its spontaneous uptake by neighboring cells and expression [12]. However, for systemic expression after for example intra-peritoneal or intra-venous injections, encapsulation of the mRNA in delivery vehicles (cationic polymers and/or liposomes) is required. Systemic expression of the protein encoded by a therapeutic mRNA can be used with the goals of vaccination (triggering of a specific immune response against the encoded protein), immunomodulation (expression of an immunomodulating protein such as a cytokine) or gene therapy (expression of a protein such as insulin). Thus safe and robust methods to deliver mRNA are required. As mentioned above, formulations of RNA using cationic polymers or liposomes are associated to pharmaceutical and toxicity issues.
In view of these drawbacks, it would be highly desirable to have an mRNA composition that could result in systemic transfection of cells without the use of transfection polymers.
The solution to the above technical problem is provided by the embodiments of the present invention as defined in the claims.
Gene Interference RNA (Antisense RNA, siRNA or miRNA)
Single stranded antisense RNA, short interfering RNA (siRNA) in the form of a duplex of complementary synthetic oligonucleotides and micro RNA (miRNA) in the form of structured stem-loop RNA molecules, can target specifically (based on its sequence) a mRNA and block its translation and/or induce its cleavage that renders it dysfunctional. Antagomirs are oligonucleotides antisense to miRNA and can block miRNA's function. “Antisense” will thus stand here for anti-mRNA as well as anti-miRNA oligonucleotides. In particular, in vitro produced siRNAs are seen as very powerful tools for therapeutic intervention for achieving the specific degradation of “pathogenic mRNA”, e.g. viral mRNA or oncogene mRNA. Since gene interference requires their intracellular localization, antisense and siRNA are formulated with polymers as above (PEI, cationic proteins, cationic sugars, liposomes, etc.) in order to be capable of efficiently penetrating cells after local (e.g. skin) or systemic (e.g. intravenous) injection. As mentioned above, formulations of RNA using cationic polymers or liposomes are associated with pharmaceutical and toxicity issues. In view of these drawbacks, it would be highly desirable to have antisense or siRNA formulations that could result in systemic transfection of cells without the use of transfection polymers.
The solution to the above technical problem is provided by the embodiments of the present invention as defined in the claims.